The present invention relates to G-CSF conjugates. The present invention also relates to a method of preparation of G-CSF conjugates with a 1:1 molar ratio between biocompatible polymer and G-CSF bonded through a thiol group of a cysteine residue in G-CSF by site-specific modification.
A variety of attempts have been made to increase the bioavailability of biologically active materials such as biologically active proteins or polypeptides, and/or extend the in vivo half-life of biologically active materials by linking them with biocompatible polymers. Conjugation of biologically active materials to biocompatible polymers may afford great advantages when they are applied in vivo and in vitro. When being covalently bonded to biocompatible polymers, biologically active materials may exhibit modified surface properties and solubility, and thus may be increased in solubility within water or organic solvents. Further, the presence of biocompatible polymers may make the proteins and/or polypeptides conjugated to them more stable, increase biocompatibility of the proteins and reduce immune response in vivo, as well as reduce the clearance rate of the proteins by the intestine, the kidney, the spleen, and/or the liver.
The most common conjugation method for reacting the proteins or polypeptides with biocompatible polymers such as PEG is achieved by reacting activated PEG to amino residues, such as lysine residues and N-termini, of proteins or peptides.
At this time, one of the hydroxyl groups of PEG is substituted with a methyl ether group while the other hydroxyl group is bonded to an electrophilic functional group to activate PEG. These methods are well known in many patents and publications.
U.S. Pat. No. 4,179,337 discloses a biologically active, substantially non-immunogenic water-soluble polypeptide composition comprising a biologically active polypeptide coupled with a coupling agent to at least one substantially linear polymer having a molecular weight of between about 500 to about 20,000 daltons selected from the group consisting of polyethylene glycol (PEG) and polypropylene glycol (PPG) wherein the polymer is unsubstituted or substituted by alkoxy or alkyl groups, wherein said alkoxy or alkyl group consists of 5 or fewer carbon atoms.
According to above-mentioned US patent, the polypeptide composition is prepared by reacting terminal carbon atoms bearing hydroxyl groups with PEG or PPG by using a coupling agent to provide an activated polymer containing a reactive terminal group, and coupling said reactive terminal group of the activated polymer to an amine group, lysine group or N-terminal group of a biologically active proteins or polypeptides. Thusly bonded PEG or PPG serves to prevent the activity of the polypeptide from being reduced.
However, this method resulted in conjugation of many PEGs to each of proteins or polypeptides, and thus, the biological activity of proteins or polypeptides was decreased. Therefore, the conjugates required purification by Reverse Phase Chromatography to obtain conjugates in which the proper number of PEGs were attached to each of proteins or polypeptides.
U.S. Pat. No. 4,301,144 discloses the hemoglobin modified by conjugating hemoglobin with polyalkylene glycol or its derivatives. It is described therein that modified hemoglobin is increased in retention time in the body, while retaining nearly the same oxygen carrying potential as native hemoglobin.
Various proteins are reported to show extended half-lives and reduced immunogenicity in plasma when being conjugated with PEG (Abuchowski et al., Cancer Biochem. Biophys., 7, 175-186, 1984).
U.S. Pat. No. 5,951,974 and Algranati et al (Hepatology, 40 (suppl), 190A, 1999) describe that PEGylation of alpha interferon with PEG12000 as well as branched PEG40000 decreases the clearance rate of alpha interferon, to enable once-weekly subcutaneous injection, instead of 3 times a week injection for native interferon.
Davis et al (Lancet, 2, 281-283, 1981) demonstrated that uricase-PEG conjugates showed increased in vivo half-life and showed reduced side effects during the metabolism of uric acid.
Colony Stimulating Factor (CSF), an acidic glycoprotein, is an important factor for survival, proliferation, differentiation of Hematopoietic Progenitor Cells (Burgess, A. W. and Metcalf, D, Blood, 56, 947-958, 1980, The hematopoietic colony stimulating factors, Elservier, Amsterdam, 1984). G-CSF has been known as a factor stimulating differentiation and proliferation of bone marrow precursor cells to granulocytes (Nicola, N. A., Metcalf, D., Matsumoto, M. and Johnson, G. R., J. Biol. Chem., 258, 9017-9023, 1983). Since mass production of CSF, which stimulates and control the proliferation and differentiation of the white blood cells, neutrophils and macrophages, has been achieved possible by using recombinant DNA technology, it is recognized that therapeutic use of G-CSF will be effective to prevent myelosuppression or enhance the recovery of bone marrow after treatment with anticancer agents. Therefore, many attempts have been made to obtain the several benefits achievable by using biocompatible polymers such as PEG to modify G-CSF.
For example, G-CSF can be reacted with methoxy PEG carboxymethyl-N-hydroxy succinimidyl ester to produce the modified G-CSF-polymer conjugates and the unstable linker of the product has been removed by treatment with 2 moles of hydroxylamine (pH7.3) followed by decreasing the pH to 3.5 (Kinstler et al., 1996, Pharmaceutical Res. 13(7): 996-1002).
Also, Niven et al (J. of Contr., Rel. 32, 177-189, 1994) demonstrated PEG conjugation of recombinant human granulocyte-colony stimulating factor (hereinafter, referred to as rhG-CSF) showed a more intense and extended white blood cell response relative to rhG-CSF alone. EP 0,401,384 describes the methods and materials to conjugate PEG to G-CSF and EP 0,473,268 describes the G-CSF conjugates between G-CSF analogs and water-soluble polymer, or PEG, via covalent bonding.
However, the biological activity of proteins has been reduced by conjugation of proteins and polymers through amine groups of proteins as described above. It is also problematic in terms of producing heterogeneous mixtures having a few PEGs attached to different numbers or sites of proteins. Therefore, an additional purification process is necessary to obtain PEG-protein conjugates having the same number of PEGs attached, the same attachment site, or homogeneous PEG-protein conjugates, and thus, this results in very low yield of final products.
The alternative to these problems is to conjugate biocompatible polymers to certain residues such as His, Trp, Asp, Glu of proteins or polypeptides, while retaining the biological activity of proteins or polypeptides. However, these residues are not suitable for conjugation with polymers since they are generally located in or near the active sites, or not exposed at the surface of proteins. Also, the numbers of these residues in proteins are too low to be conjugated with sufficient numbers of polymers to extend the plasma half-life of proteins significantly, and the reaction condition is not site-specific, as well as the reaction condition for site-specific conjugation being too harsh, thus decreasing the biological activity of proteins.
The site-specific modification of proteins with polymers through a thiol group of a cysteine residue is the one method to overcome this problem. In general, only a few proteins possess the accessible thiol group(s) and several methods of site-specific conjugation have been suggested to conjugate PEGs to natural thiol groups of cysteines or substituted cysteines by genetic engineering method.
For example, in order to conjugate PEG to thiol groups of cysteines in proteins or polypeptides, it is known that a stable symmetric disulfide is obtained by using PEG-ortho-pyridyl-disulfide (Woghiren et al. Bioconjugate Chem 1993, 50:75-80), and an activated double bond is reacted with thiol groups by the Michael reaction using PEG-maleimide. (Ishii et al., Biophys J. 1986, 50:75-80). Also, Shaw et al. (U.S. Pat. No. 5,166,322) describes the preparation of PEG-IL-3 by reacting the activated PEG, or sulfo-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate activated PEG with the thiol groups of cysteine residues substituted for a lysine residue.
Also, Katre et al. (U.S. Pat. No. 5,206,344) describes the conjugation of an IL-2 mutant having a substituted cysteine at a certain site of IL-2, with activated PEG, or maleimido-6-amidocaproyl ester activated PEG4000. Rich et al. (Rich, D., et. al., J. Med. Chem. 18, 1004, 1975) reported the chemical modification of thiol groups of proteins using gamma-maleimido butyric acid and beta-maleimido propionic acid.
Braxton et al. (U.S. Pat. No. 5,766,897) describes PEG-cysteine protein conjugates, and especially the detailed method for covalent conjugation of cysteine groups of protease Nexin-1 with mPEG-maleimide.
WO 01/87925 describes a method of obtaining refolded soluble proteins having at least one free cysteine residue from insoluble or aggregated proteins, and exemplifies cysteine-reactive PEGs and PEGylated proteins, for example PEGylated G-CSF formed from cysteine-reactive PEG as examples of reactive cysteine residues. However, in relation with cysteine-PEGylation of wild type G-CSF, the PCT publication states clearly that wild type G-CSF was not PEGylated under the reaction conditions of this publication, as specifically described in Example 10.
These methods mentioned previously relate to methods for conjugating biocompatible polymers to thiol groups of cysteine residues of proteins or polypeptides, or relate to methods for increasing the biological activity of proteins conjugated with a number of biocompatible polymers. However, these works have not reported that 1:1 conjugates between a biocompatible polymer and a thiol group of a cysteine residue of G-CSF unexpectedly showed the significantly improved effect, for example, improved stability and biological activity.
Although G-CSF contains 5 cysteine residues, the present inventors found that G-CSF was bound to a biocompatible polymer stoichiometrically at a 1:1 molar ratio under generally known reaction condition through a thiol group of a cysteine residue and these conjugates had more improved stability and biological activity compared to G-CSF conjugates with several PEGs attached.
The G-CSF conjugates of the present invention are obtained as protein-polymer conjugates at a 1:1 molar ratio and thus provide a homogeneous product which may have benefits in the aspect of clinical study and better stability profiles than the conjugates bonded through amine groups of G-CSF. Therefore, G-CSF conjugates of the present invention have an additional advantage that a further purification process using the preparative column is not necessary.